Dermal filler composed of macroporous chitosan microbeads and cross-linked hyaluronic acid

ABSTRACT

A biocompatible, degradable dermal filler system is composed of unmodified macroporous chitosan microbeads dispersed uniformly in a continuous phase composed of cross-linked hyaluronic acid gel particles and unmodified hyaluronic acid.

FIELD OF THE INVENTION

The present invention pertains to biocompatible compositions for softtissue augmentation, more specifically to a dermal filler containingabsorbable chitosan microbeads consisting of pure chitosan, or modifiedby a chemical crosslinker. The chitosan microbeads are suspended in amatrix of cross-linked hyaluronic gel particles, wherein the microbeadscomprise a slowly-resorbing component in an augmentation system designedto provide both short-term and long-term augmentation for the treatmentof cosmetic or medical conditions, which require a biocompatiblespace-occupying substance.

Applications include the treatment of facial wrinkles or folds, ortreatment of a wasting medical condition, such as lipoatrophy. Indifferent embodiments of the invention, a pharmaceutical ingredient maybe included, for the control of injection pain. Furthermore, the presentinvention pertains to a process for preparing the chitosan microbeads,and for combining them with cross-linked hyaluronic acid gels, in anaugmentation system. The combined system also comprises the formation ofa polyelectrolyte complex at the surface of the microbeads, throughinteraction of HA and chitosan, which is important in regulating theabsorption of the microbeads. The macroporous nature of the beads, alongwith the biocompatibility of chitosan provide a scaffold for theproliferation of fibroblasts and the subsequent deposition of naturalcollagen.

The present invention also pertains to specific methods of producing themacroporous chitosan microbeads. Both emulsion/solvent evaporation, andemulsion/neutralization methods have been developed. The evaporationmethod leads to microbeads with large pores, on the scale of themicrobead itself, providing an excellent scaffold for cell growth andnatural collagen deposition.

BACKGROUND OF THE INVENTION:

Dermal fillers have been used to offset the effects of aging on theskin, by smoothing soft tissue defects like nasolabial folds andmarionette lines as well as more substantial augmentation such assmoothing hollow cheeks resulting from lipoatrophy, or enhancing thefullness of lips. Fillers must be able to satisfy a number of needs,depending on the type of defect which needs to be corrected.

Currently the predominant formulation of dermal fillers is based onchemically-modified Hyaluronic Acid (HA), which has largely supplantedearlier products based on bovine collagen, which suffered from poordurability and the need for an allergy test. Concerns regarding bovinespongiform encephalopathy (BSE) disease also played a role in thedeclining popularity of the collagen fillers.

Chemical cross-linking of HA is necessary in order to increase thedurability of the implant, given that unmodified HA has a half-life inthe dermis of only a day or two. Products of this type are Restylane®,Perlane®, Puragen®, the Juvéderm® family, the Esthélis® family, and theRevanesse® family. Notably, all of these fillers are based oncross-linking via the same chemical compound, ButaneDiolDiglycidylEther(BDDE), with differences in the products arising from the details of themanufacturing processes used, including process steps after chemicalmodification to prepare a sterile, injectable gel, delivered in apre-filled syringe.

A filler which can also lead to enhanced deposition of natural collagenis very desirable, as the effects would be long-lived and completelynatural. There is also a need for a type of filler, which can providesignificant enhancement, with long duration as well as natural collagendeposition. This should be accomplished without inducing an ongoinginflammatory reaction. To accomplish this, a delicate balance isrequired between the rate of absorption of the implant and stimulationof the skin to produce collagen, avoiding overstimulation leading toinflammation.

The inclusion of solid, biocompatible microbeads in a biocompatiblecarrier has been one approach to meeting this need for long duration andnatural collagen deposition. The carrier is usually absorbed in arelatively short period. However, it has proven to be difficult toachieve the correct balance, and these more-permanent fillers have hadissues regarding excessive tissue reaction after implantation ortoo-rapid absorption. These treatment can also be difficult to reverse,if it is deemed necessary, by injection of natural enzymes. This can bedone with the HA-based fillers (hyaluronidase). An ideal combination ofdurability and stimulation of collagen deposition, with low inflammationhas yet to be achieved.

Some materials that have been used for these microbeads arepolymethylmethacrylate (PMMA), described in U.S. Pat. No. 5,344,452,polylactides (polylactic acid or PLA), described in U.S. Pat. No.6,716,251, and calcium hydroxylapatite, described in U.S. Pat. No.7,060,287. Recent patents describing the potential use ofpolycaprolactone or polydioxanone microbeads, are U.S. Pat. Nos.7,964,211, and 9,119,902. All of these biomaterials have been usedpreviously in other medical applications: PMMA in intraocular lenses andbone cement, PLA in bone pins and screws, polycaprolactone in absorbablesutures and drug delivery devices, and hydroxylapatite as a bone fillerand contrast agent. These materials were then adapted for use in adermal filler by forming microbeads from the raw material. Durability ofthese fillers ranges from months, to several years to permanent.

Typically these microbeads are sized between 40 and 150 μm. If thespheres are too small they can elicit a reaction from macrophages, whichwill attempt to engulf the particles. If the spheres are too large, theycannot be injected with the fine needles that are used in dermal fillingprocedures, and they may be palpable under the skin.

Chitosan is a naturally-occurring biopolymer, a linear polysaccharidecomposed of randomly distributed β-(1-4)-linked D-glucosamine andN-acetyl-D-glucosamine units. A related form is chitin, which is simplya linear chain of β-(1-4)-linked N-acetyl-D-glucosamine, and from whichchitosan can be derived. Chitin is the primary component of arthropodexoskeletons and is the second most abundant naturally-occurringbiopolymer after cellulose. Chitin is also the source for mostcommercial chitosan.

Both chitin and chitosan have a number of industrial uses, e.g. foodprocessing and preservation, waste-water treatment, and chromatography.In the medical field chitosan has drawn attention due primarily to itsbiocompatibility, biodegradability, and antimicrobial properties.Chitosan has been extensively studied for tissue engineering and drugdelivery applications. The ability to form microspheres as well aspolyelectrolyte complexes with anionic biopolymers like alginate,carboxymethylcellulose, and hyaluronic acid is important in some ofthese proposed applications. The mucoadhesive properties of chitosanhave also been studied for use in nasal and ocular drug delivery. Theability of chitosan and chitin to speed the wound healing process hasbeen known for decades and commercial products have been marketed. Theantimicrobial property as well as the promotion of healing arebeneficial in a dermal filler application as well.

Chitin and chitosan have a cellulose-like molecular structure. Chitin,due to the stability of its crystalline form, is insoluble in water. Therandom arrangement of acetylated and deacetylated units along thechitosan chain and the possibility for protonation of the glucosamineunits, means that chitosan is soluble in weakly acidic aqueoussolutions, where it exists in the form of a polycation. The precise pHat which chitosan dissolves depends on the molecular weight, the degreeof deactylation (proportion of glucosamine units), and the degree ofrandomness of the arrangement of the two units along the chain. Chitosanwith a partial block-like structure is produced in some processes, andthis material is less soluble in water.

A commonly noted drawback of chitosan is that it is generally insolubleat a pH above approximately 6.5, which is slightly lower thanphysiological pH. This has hindered its adoption in medicine for someapplications. Another issue has been the difficulty in sourcinghigh-quality raw material. A great deal of research has been directedtowards methods of increasing the aqueous solubility of chitosan byderivitization, choice of counterion, or by meticulous control ofmolecular weight, degree of deacetylation, and the distribution of baseunits along the linear chain. Generally, the goal has been to develophydrogels that could be used, for example, in drug delivery or tissueengineering.

Objects of the Invention:

The objects of the present invention are to provide a composition fordermal filler applications that can be injected with a fine-gauge needleand provide 1) an immediate augmentation effect from a proven HA-basedcomposition, which additionally includes a chitosan microbead component,2) provide long-lasting augmentation from the chitosan microbeads, asthe HA gel is absorbed, 3) provide for the deposition of naturalcollagen, around and inside the microbeads, as they are slowly absorbed,with 4) no excessive inflammatory reaction, or formation of granulomas.

These objectives constitute ideal properties for a long-lasting tissueaugmentation product, in particular for a cosmetic dermal filler, wherelonger duration of the correction is desired.

All four objectives have been demonstrated for our composition in ayear-long rat implantation study, which included three implementationsof our invention, with high-density and low-density macroporousunmodified chitosan microbeads as well as a formulation containingchemically-crosslinked chitosan microbeads.

In the present invention, the drawback that chitosan does not dissolvereadily in tissue and remains a solid after implantation, becomes anadvantage. Simple, unmodified chitosan microbeads can be used in afiller application, and no additional chemical stabilization of thisbiocompatible polysaccharide is necessary. However, the borderlineinsolubility of chitosan at physiological pH means that the material isnot inert, and will be slowly degraded at the implantation site. Themonomeric components of chitosan are N-acetyl glucosamine andglucosamine. Both of these monosaccharides are naturally occurring inthe human body, and are necessarily biocompatible. Our primary inventioncomprises the use of unmodified chitosan microbeads combined withcross-linked hyaluronic acid gel in a dermal filler. The inventionincludes the specific process by which the microbeads are formed,leading to a macroporous structure, as well as the discovery that anHA-Chitosan polyelectrolyte complex forms at the surface of themicrobeads in our formulation and significantly enhances the durabilityof the microbeads.

We also claim an additional embodiment of our invention, which uses achemical cross-linker to further stabilize the chitosan microbeadcomponent and extend the duration of the implant, for applications wherea more permanent augmentation is desired. We claim the use of unmodifiedmicrobeads from either of our productions processes, and adding the stepof chemical cross-linking between sites on the chitosan polymer chain inthe previously-formed microbead.

Given the existence of primary amines (glucosamine) along the chitosanchain, there are many methods that can be employed. Amines will reactwith a wide variety of bifunctional cross-linkers. For one skilled inthe art, these include biisothiocyanates forming isothiourea bonds,biisocyanates forming isourea bonds, biazides forming amide bonds,dialdehydes forming secondary amines after reduction, dicarboxylicacids, esterified by N-hydroxysuccinimide (NHS), or sulfo-NHS, formingamide bonds, dicarboxylic acids in combination with carbodiimidesforming amide bonds, and bisepoxides forming secondary amine bonds.

In the present invention, bisepoxides are preferred, and BDDE is mostpreferred, due to its widespread use in stabilizing hyaluronic acid fordermal filler applications and the established safety profile it has inthis product area.

SUMMARY OF THE INVENTION

The present invention is directed to a dermal filler comprising acombination of biocompatible, absorbable, macroporous chitosanmicrobeads dispersed in a gel particle phase of cross-linked hyaluronicacid. Also described are methods for making such microbeads as well asstandard methods for producing the cross-linked HA gel phase.

The production of chitosan microbeads (or microspheres) has beenconsidered by a number of researchers. Standard techniques have beenemployed. Generally droplets of a chitosan solution are formed, andhardened into microbeads by different methods.

Aqueous chitosan solution droplets can be formed and hardened intomicrobeads in a spray dryer. The solution is forced through a nozzle orexpelled by a spinning disk, into a heated gas phase, wherein the waterfrom the droplet evaporates and a microbead is formed. Difficultiesarise in controlling the size and shape of the microbeads.

A simple method to produce chitosan beads is by extrusion throughnozzles or needles of an acidic chitosan solution into an alkalinesolution, called ionotropic gelation, or more simply, neutralization.Chitosan is a polycation, so chitosan solution droplets can also beextruded into a solution of a polyanion like triphosphate, for physicalcross-linking. After extrusion the increase in the pH or the presence ofthe polyanion lowers the solubility of the chitosan, initially forming agel particle from the droplet and finally solid beads. Generally inthese methods the beads are relatively large and shapes are often poorlycontrolled. Methods for forming small, uniform-sized microbeads, such asinjection of droplets from microfluidic devices offer good control ofdispersity, but are impractical due to the low production rates that areachievable, particularly if the goal is to produce small droplets, withmicrobeads with diameters on the order of 100 μm.

A more practical means of producing large numbers of small microbeads isto produce the droplets by emulsification of a chitosan solution in anon-aqueous phase. The hardening of the droplets into microbeads can beaccomplished by changes in pH or the addition of chemical or physicalcross-linking agents. In our initial experience, addition of an alkalinesolution to the emulsion resulted in rapid gelation at the surface, withlow-density microbeads, that collapse on drying with poor control overthe final shape. After drying, the microbeads were reduced in size anddid not absorb water well, due to a high degree of crystallinity in thesolid. However, a modified emulsion/neutralization method based ondiscoveries we made during the development of our emulsion/solventevaporation method was more successful and is described below as Method2.

Another approach based on emulsification is to choose an ‘oil phase’with a non-zero solubility for water, but no solubility for chitosan.One example of this method, described by Baimark and Srisuwan (2013) isto use ethyl acetate as the ‘oil’ in a W/O emulsion of a chitosansolution. After the emulsion is formed water is drawn out of thedroplets into the organic phase in which it is partially soluble, andeventually solid microbeads can be obtained. The drawback to thisapproach is the need to use very large amounts of the organic solvent.Some fraction of water must dissolve into the organic phase in thisapproach, but the concentration is typically very low for a system thatmust also form two distinct phases. As a result this method requireslarge amounts of organic solvent relative to the mass of microbeads thatcan be produced. Given these limitations solvent diffusion was notpursued as a suitable commercial method, for manufacture of ourmicrobeads.

Method 1: Another emulsion-based method which can produce smallmicrobeads with acceptable control over the size distribution areemulsion/solvent-evaporation methods. In this approach a stable emulsionis produced with controlled-size droplets. After an appropriate changein conditions, such as elevating the temperature, a loss of solventoccurs in the droplets, until a solid microbead is formed. This methodrequires evaporation of water through an immiscible non-aqueous phase.This can be done slowly, leading to a more spherical shape and smoothsurface structure for the microbeads. This is the favored approach weemploy in our invention to form the unmodified chitosan microbeads,which is also the source material for the cross-linked version of ourmicrobeads.

The emulsion from which the microbeads are produced is formed in twostages. First, the initial emulsion is obtained by homogenizing anaqueous acidic chitosan solution in an oil phase in the presence ofemulsifier, to form an initial W/O emulsion. This primary emulsion isthen stabilized by dilution with additional oil to form the secondaryemulsion. Afterwards the chitosan microbeads are formed from thedroplets in the secondary emulsion by diffusion of water through the oilphase and evaporation at the surface and by agglomeration of the primaryparticles into the microbeads.

A unique aspect of our process is the formation of anOil-in-Water-in-Oil, or 0/W/0 emulsion, in the primary emulsion, due tothe high W/O ratio possible with the choice of castor oil for theprimary emulsion. During the drying phase in the secondary emulsion theprimary gel particles aggregate to form the final microbead, but thesecarry with them a portion of the castor oil phase, which coalesces intolarge oil droplets in the microbead as it forms (FIG. 2). After thewashing step removes the oil, the resulting voids are the source of thelarge pores in the final microbead.

It is the formation of this special phase that results in the largemacroporous structure of our beads after removal of the entrapped oil,and is an important aspect of the invention. In the final step, themicrobeads are washed with an organic solvent, removing the oil phase,and leaving a macroporous structure in the resulting dried microbead.The macroporosity is a key feature, which assists in controlling thedegradation of the microbeads and allows for the ingress andproliferation of fibroblasts leading to slow replacement of themicrobead by natural collagen deposition, after implantation in tissue.Evidence from our rat implantation study supports this conclusion asshown in FIGS. 8 and 9.

Some of the process parameters that affect the final microbead productare the composition of the aqueous and oil phases, the molecular weightof the chitosan and its degree of deacetylation, the mixing geometry,speed, and time, the water/oil phase ratio, the evaporation temperatureand even the external conditions of surface to volume ratio, humidityand air flow. These can affect not only the size and shape of themicrobeads, but the surface smoothness and porosity. Conditions employedin our invention are described and claimed as part of the invention inthe detailed description and examples below.

As noted, the microbeads are cleaned, neutralized, and dried, obtaininghighly purified macroporous microbeads appropriate for inclusion in aninjectable product. The ‘near solubility’ of the microbead isdemonstrated by the observation that it swells significantly, byapproximately 50%, but does not dissolve when equilibrated inphosphate-buffered saline at a pH of 7.0. The microbeads prepared by ourprocess flow well, and can be uniformly dispersed into the cross-linkedHA gel particle phase at the desired concentration, without damage.After formulation the microbeads produced in our process can alsowithstand automatic filling into syringes and terminal sterilization bymoist heat in an autoclave. The method is described in detail below asMethod 1.

Method 2: During the development of the solvent evaporation method thediscoveries we made concerning the properties of different oils allowedus to develop an improved approach to the emulsion/neutralizationmethod. Briefly a chitosan solution can be dispersed in castor oil, withor without the addition of an emulsifier.

In previous attempts by us to use the neutralization method, aqueousbases such as a sodium hydroxide solution were employed. This formed asecond water phase, with droplets of sodium hydroxide solution. As thesecame in contact with the chitosan solution the pH would be lowered andthe droplets hardened. However, this produced a very irregulardistribution of microbeads, with many large agglomerates.

In the method described here, the droplets are neutralized by additionof a base with significant solubility in the castor oil phase. This isan important discovery, as we find that it leads to microbeads withessentially spherical shapes and good control over the sizedistribution. The fact that the base can reach the aqueous droplets ofchitosan solution by diffusion through the oil phase is the key to thisimprovement. We find that lowering the pH in this way causessolidification in as little as 20 minutes.

Dilution with solvents miscible with both water and castor oil, permitsisolation of these microbeads by filtration or sedimentation. Thismethod also produces porous chitosan microbeads, with a somewhat broadersize distribution than the solvent evaporation method. This alternativemethod for production of chitosan microbeads is described in detailbelow as Method 2.

DETAILED DESCRIPTION OF THE INVENTION Chitosan Bead Formation—Method 1:Emulsion/Solvent Evaporation

Many factors affect the final state of the chitosan microbeads formed inour process: the molecular weight and degree of deacetylation of thechitosan; the type of acid and the concentration used in the chitosansolution; the chitosan concentration; the composition and resultinghydrophobicity and viscosity of the oil phase; the type andconcentration of the emulsifier; the O/W ratio in the primary andsecondary emulsions, the mixing apparatus used; and the evaporationconditions, including temperature and geometry.

Chitosan Raw Material:

Choice of raw material is important in the production of high qualitychitosan microbeads. As noted above, two factors are important indetermining the physical properties of chitosan, the molecular weightand the degree of deacetylation. The distribution along the chain canalso be important but this is not determined by suppliers.

In our method, to be described here in detail, we have discovered thatincreasing molecular weight tends to produce more spherical microbeads,with a smoother surface, and a narrower size distribution. We have alsodetermined that the degree of deacetylation mainly affects thecrystallinity of the microbeads, which in turn affects the degree ofswelling when the dried beads are rehydrated. The crystallinity willalso affect the rate at which degradation will occur in-vivo.

Many suppliers provide only a wide specification range for molecularweight and degree of deacetylation. We have determined that, in ourmethod, molecular weights from 100 to 2000 kDa can be used, morepreferably from 300 to 700 kDa. A degree of deacetylation from 65 to 95%can be used, more preferably, from 80 to 90%.

Chitosan Solution:

At low pH amine groups on chitosan are protonated, increasing thesolubility in aqueous solution. Chitosan can be dissolved in a varietyof organic acids such as acetic acid, formic acid, adipic acid, ascorbicacid and lactic acid, or dilute inorganic acids such as hydrochloricacid or phosphoric acid. Any of these can be used in our method and areincluded in the invention. Preferably the solution is prepared witheither acetic acid or dilute hydrochloric acid, and most preferably withacetic acid.

The molecular weight, the degree of deacetylation, the concentration andthe pH all affect the physical properties of the chitosan solution, mostimportantly the viscosity. This affects the size of the droplets, withother conditions held constant. The concentration and size of thedroplets determines the size and porosity of the resulting chitosanmicrobeads. These factors can all be adjusted to produce a range ofmicrobead sizes, swelling characteristics, and final chitosan density inthe swelled microbeads.

For example: chitosan with a molecular weight specification of between140 kDa and 220 kDa at a concentration of 4% dissolved in 5% AcOH has aviscosity of 30.28 Pa·s, while a solution with a concentration of 4.44%in 10% AcOH has a similar viscosity of 31.25 Pa·s. We have discoveredthat those solutions with the same viscosity, produce the same particlesize in our method, however with different densities. This is an exampleof how various inputs in our process can be used to control the finalproperties of the microbeads.

Acceptable concentrations of chitosan in our method depend on themolecular weight and on the concentration of acid but are between 1% and5%, preferably between 2% and 3%. Hydrochloric acid concentrations canbe used between 0.1N and 0.2N, most preferably between 0.16N and 0.18N.Lactic acid concentrations can be used between 1 and 10%, mostpreferably between 2% and 3%. Acetic acid concentrations can rangebetween 2% and 10%, preferably between 4% and 6% and most preferably at5%.

Oil Phase Composition:

A wide variety of non-toxic oils can be used as the continuous phase inthe primary emulsion. For the highest levels of safety, preferred oilsare those listed in the FDA Inactive ingredients guide forintramuscular, intravenous, or intradermal injection. These oils havebeen used in the development of various drug products, or drug deliverysystems. These include vegetable oils, such as corn oil, soybean oil,canola oil, castor oil, sesame oil, peanut oil and almond oil, or lightmineral oil and mineral oil. The oil is not part of the finalformulation of the filler system of course, but restricting theproduction process to the use of these oils, adds to the assurance ofsafety for the final product.

Three important parameters for the oil phase that pertain to our methodare the viscosity, the polarity of the oil, and the interfacial tensionbetween the oil and aqueous phase. The aqueous chitosan solution isviscous and we have discovered that the more closely the viscosity ofthe oil phase matches the chitosan solution, the more uniform the sizedistribution of the microbeads. High interfacial tension is obtainedwith hydrophobic, non-polar oils like mineral oil, and this tends toproduce microbeads with a smooth surface and spherical shape. However,water evaporation from the droplets, which is necessary to form themicrobeads, is impractically slow when the oil continuous phase is veryhydrophobic, as there is extremely limited solubility of water in theseoils.

In addition, the low viscosity of the mineral oils relative to thechitosan solution, tends to produce microbeads with a wide sizedistribution. A wide distribution was also noted with some of thevegetable oils, such as corn oil, also due to relatively low viscosity.The low viscosity of these oils also tended to produce less stableemulsions due to increased rates of droplet coalescence, allowing lesstime for transfer to the secondary emulsion.

Castor oil was found to be an excellent choice on the basis of its highpolarity (due to the hydroxyl group on ricinoleic acid), and its highviscosity, which is a good match to the viscosity of the chitosansolutions in our method. The result is good uniformity in the sizedistribution of droplets in the primary emulsion. The high viscosityalso allows for a high W/O ratio (up to 9/10), important for commercialrates of production. We also discovered that these high ratios of W/Oled to the formation of the O/W/O multiple emulsion, critical to theformation of the macroporous structure of the final microbeads, shownfor example in FIGS. 8 and 9. The dependence of the formation of themultiple emulsion and the subsequent macroporous structure of the beadsis not obvious, and is an important part of the invention describedhere.

Water is slightly soluble in castor oil, due to its polarity, whichallows for an acceptable evaporation rate, with water moving from thedroplets to the oil and then evaporating at the surface. However thatsame polarity and slight miscibility with water, leads to a low valuefor the interfacial tension, leading in turn to poor sphericity andsmoothness for the microbeads.

Castor oil proved to be an ideal oil for the primary emulsion. Howeverthe issues noted above indicated that a mixture of castor oil andmineral oil if used for the oil phase in the secondary emulsion, mightlead to an improved local structure for the microbeads. It is in thesecondary emulsion where the evaporation/hardening of the droplets andaggregation into the chitosan microbeads occurs. However these two oils,due to their difference in polarity are not miscible. However, we havemade the discovery that oils with an intermediate polarity from the listof those oils approved for injection by the US FDA, can act assolubilizing agents between castor oil and light mineral oil. Thepreferred oil for this purpose is corn oil.

The coexistence curve for the three component system, castor oil, lightmineral oil, and corn oil, has been mapped on a ternary phase diagram attwo temperatures. This is shown in FIG. 1. We have found that asingle-phase region exists, dependent on the temperature, wherein thecombination of oils is miscible and this combination can be used as thecontinuous phase in our secondary emulsion. So, we have discovered ameans, using oils that we consider acceptable, for adjusting importantproperties of the oil phase, to optimize the production rate and qualityof the chitosan microbeads. The combination of mineral and castor oil bythe addition of a third oil (corn oil in this particular embodiment) isan important part of our process and the invention described herein.

A range of compositions for the secondary emulsion oil phase can be usedwith components of castor oil, corn oil and light mineral oil. Based ona nunber of designed experiments, our preferred composition ratio is10/20/20 for castor oil/corn oil/light mineral oil.

Emulsifier:

A variety of biologically compatible emulsifiers (FDA-approved foreither injection or transdermal application) can be used including thetwo families of sorbitol derivatives, the hydrophobic Span® family, inparticular Sorbitan Monopalmitate and Sorbitan Monooleate, and thehydrophilic Tween® family, particularly PEG-20 Sorbitan Isostearate.Castor oil derivatives such as PEG-40 Castor Oil, PEG-60 HydrogenatedCastor Oil, and Polyoxyl 35 Castor Oil can also be used, as well as theGlyceryl derivatives, Glyceryl Palmitostearate, Glyceryl Oleate,Glyceryl Trioleate, and Glyceryl Laurate, as well as the Poloxamerfamily of nonionic emulsifiers, in particular Poloxamer 188.Hydrogenated Soybean Lecithin can also be used. Most preferred is thenatural emulsifier, Lecithin.

In the primary emulsification of the chitosan solution, a range ofconcentrations of lecithin can be used, from 1% to 5%. Most preferred is2% lecithin in castor oil.

Primary Emulsification Process:

In our method, emulsification can be carried out in a simple mixingsystem. There is no necessity for turbine-style mixers, high-pressurehomogenizers (Manton-Gaulin, Microfluider®), or colloid mills, althoughthese could potentially be used.

We have however observed that mixing speed and duration can influencethe particle size, within a certain range. For example, a sampleprepared at 450 rpm for 1.5 minutes of mixing time shows a similar meanparticle size (65.8 μm) as another sample, which was prepared at 400 rpmand 2 min (63.7 μm), holding other parameters constant.

As noted, we have also made an important discovery, that by using asufficiently high W/O ratio in the primary emulsion with castor oil,that droplets of oil are also incorporated into the aqueous chitosanphase. We have at that stage an O/W/O multiple emulsion. Afteraggregation of primary gel particles to form solid microbeads, thecoalesced oil droplets are later removed in the final washing step,leaving behind macroporous chitosan microbeads. The pore structureassists in controlling the degradation of the microbeads and allows forthe ingress and proliferation of fibroblasts leading to slow replacementof the microbead by natural collagen deposition.

Secondary Emulsion and Evaporation:

A secondary emulsion is prepared by dilution of the primary emulsion inan excess of the oil phase used in the primary emulsion. By forming adilute suspension of the aqueous chitosan solution droplets in an excessof the oil phase, we control the stability for the evaporation/hardeningstep, by reducing the frequency of droplet-droplet collisions. Under theconditions, wherein lecithin is used at 2% in castor oil for the primaryemulsion and the W/O ratio is 0.9, we have discovered that no additionallecithin is required in the oil fraction of the secondary emulsion forthe preferred final composition of 10/20/20 for castor oil/cornoil/light mineral oil, in order to maintain the stability of thedroplets during evaporation. We have also discovered that continued mildstirring of the secondary emulsion is the best condition to maintainstability during the evaporation process.

The evaporation temperature, along with the geometry of the hardeningtank, and the external conditions of air flow and humidity all affectthe evaporation rate. Temperature is important in several respects.Although higher temperatures shorten the drying/hardening time, highertemperatures can also destabilize the emulsion. We have found thattemperatures for the secondary emulsion between 20° C. and 40° C. can beused in the evaporation process, more preferably between 20° C. and 30°C., and most preferably between 26° C. and 28° C. In particular theseconditions are ideal for the preferred oil composition of 10/20/20,castor oil/corn oil/light mineral oil.

As noted in the brief description the formation of the microbeads in thesecondary emulsion is a complex process. Incorporated into the primaryaqueous droplets are some droplets of the castor oil phase. Asevaporation begins and the droplets develop a gel-like nature there isalso some aggregation of the gel particles, and finally the formation ofsolid microbeads, which still incorporate oil. The contraction of themicrobead on drying leads to the existence of relatively large pores inthe structure of the bead. The removal of these pores in the washingprocess leads to the final macroporous structure of the chitosanmicrobeads, evident in FIGS. 8 and 9.

Washing and Neutralization:

Washing: The secondary emulsion turns clear after continuous stirringfor 10-24 hours, indicating the microbeads are solidified. The oil phasecontaining microspheres is then transferred to a centrifugation tube andcentrifuged at 1000 G for 1 min, the supernatant oil is decanted, andthe microspheres at the bottom are then washed twice with ethyl acetateand twice with n-hexane by vortex mixing, followed by centrifugation, toremove the residual oil. Trace amounts of lecithin are removed bywashing twice with ethanol, and then the clean beads are allowed to dryin air.

Neutralization: Chitosan beads as obtained were first soaked insaturated Na₂CO₃ solution for 10 minutes, then washed with DI waterseveral times to produce the protonated form of chitosan. They werefurther subjected to washing with PBS, until a neutral pH was achieved.After the removal of surface salt by rinsing with DI H₂O, the beads weredehydrated with ethanol and air dried. The final neutral beads werestored in sealed vial at room temperature prior to use.

Final Mass Density:

After swelling in phosphate-buffered saline, the beads swell byapproximately 50%, and the final mass concentration of chitosan in themicrobeads ranges from 8% to 20% but preferably between 10% and 16%. Forunmodified chitosan microbeads, the densities, 9.9% and 16.2%, were usedin the rat implant study. The details of the process for preparing thesemicrobeads are described in Examples 1, and 2. The results of theimplant study are discussed below.

Size Distribution:

Typical size distributions are shown in FIG. 1, wherein the mean size isapproximately 95 μm with a standard deviation of 20 μm.

Cross-Linking of Chitosan Microbeads:

Our invention demonstrates the feasibility and desirability of usingumodified chitosan microbeads in a dermal filler system in combinationwith cross-linked hyaluronic acid gel particles. However, we have alsodiscovered a simple method, using BDDE, a chemical cross-linkerwell-known in the art of HA-based dermal fillers, to produce chemicallycross-linked chitosan microbeads, with the same macroporous structure asthe unmodified beads described above. This is accomplished by usingmicrobeads from Method 1. The cross-linking occurs under basicconditions, so the microbead structure is maintained. An example of thisprocess is provided in Example 3.

Although the microbeads are not solubilized undert the basic conditionsused during the cross-linking, the reaction still occurs. This discoveryis also part of the invention described herein. The existence ofchemical cross-links in these modified microbeads is demonstrated by alack of solubility in 5% acetic acid solution, which will readilydissolve the unmodified microbeads used as source material in thecross-linking reaction. Cross-linked microbeads with a density of 14.7%were used in the rat implant study and those results are also discussedbelow.

Chitosan Bead Formation—Method 2: Emulsion/Neutralization Hardening

Given the unique characteristics we had discovered for a W/O emulsion ofchitosan solution in castor oil, we examined again theemulsion/neutralization process for forming microbeads from an emulsionof an acidic chitosan solution dispersed in a castor oil continuousphase. We were surprised that these results were superior to thoseachieved using other emulsion systems. We also made discoveriesregarding the types of base that must be used for neutralization, whichwere dependent on their solubility in the castor oil phase.

Basic steps in this method involve the dispersion of an aqueous acidicchitosan solution in an oil phase, most preferably castor oil, followedby the addition of a base to precipitate the solution droplets into agel/solid phase, followed by dilution with an oil-miscible low-viscosityorganic solvent to allow for separation of the microbeads bycentrifugation or filtration. The collected microbeads are then washedand dried, or washed and suspended in phosphate-buffered saline.

Surprisingly we discovered that chitosan microbeads could be producedfrom a primary emulsion in castor oil, either with an emulsifier as inour solvent evaporation method, or in the absence of an emulsifier,depending on the desired size of the microbeads. We discovered thatcastor oil intrinsically acts as a weak emulsifier, due to the hydroxylgroup on the aliphatic chain of ricinoleic acid, the primary fatty acidin castor oil. Surprisingly, the stability of the dispersion isadequate, given that our neutralization method is very rapid. Anemulsifier, such as lecithin does have the effect of reducing thedroplet size, if other conditions such as W/O ratio, mixing apparatus,and temperature remain constant, but is not essential.

We discovered that ordinary aqueous bases like sodium hydroxide solutionproduced irregular, poorly-controlled microbeads, often with largeagglomerates. We subsequently discovered that if the base hassignificant solubility in the castor oil phase, superior results, withthe formation of spherical beads with adequate size control arepossible. This is true even if an aqueous solution of the base is used,as long as the base itself has solubility in the castor oil.

A variety of bases can be used for neutralization as long as there issome solubility in the castor oil phase, or other oil phase used. Aminebases can be used, such as the aliphatic primary amines, methylamine,ethylamine, propylamine, isopropylamine, and other members of thisfamily; secondary amines such as dimethylamine, and tertiary amines suchas triethylamine can also be used. Amino alcohols such as ethanolamine,triethanolamine, and tris(hydroxymethyl)aminomethane are also effectiveneutralizers. Ammonia gas, NH₃ can be used to neutralize the droplets bybubbling the gas through the emulsion, or more conveniently, aconcentrated NH₄OH solution can be added to the emulsion. As notedabove, although the aqueous NH₄OH solution forms a separate dispersedphase in the emulsion, we have discovered that neutralization of thechitosan droplets occurs at a good rate by diffusion of ammonia from theammonium hydroxide solution through the oil phase to the droplets. Themicrobeads which are formed in this process are also porous with adensity comparable to those obtained in the solvent evaporation methoddescribed previously.

After neutralization the microbeads can be separated from the oil phaseby the addition of a oil-compatible diluent, to lower the viscosity ofthe continuous phase and allow for separation of the microbeads from thecontinuous phase, either by filtration or centrifugation. A variety ofcommon organic solvents can be used as a diluent, if they are misciblewith castor oil. Particularly useful are solvents, which are alsomiscible with water, and relatively volatile, to make removal of thesolvent simple. Some examples are acetone or acetonitrile, or alcoholssuch as methanol, ethanol, or propanol. Most preferred is ethanol forreasons of toxicity as well as ease of removal.

The same solvent can then be used to wash the microbeads and remove anyresidual oil. Resuspending the microbeads in PBS, tends to maintain thespherical shape, until needed for incorporation into the filler, and wehave found it superior to complete drying of the microbeads, although wehave also followed this procedure and successfully obtained microbeads.

Other discoveries disclosed herein, that are part of the invention isthe observation that in this simple method, chitosan solution must beadded to the oil, either drop wise or by injection, while stirring, toavoid excessive encapsulation of oil in the chitosan microbeads anddisruption of the microbead structure; and the emulsion must also befully neutralized by the base before workup, to avoid agglomeration andfragmentation of the microbeads.

It was discovered that if the viscosity of the chitosan solution was 2or 3 times that of castor oil, but not higher, microbeads of the desiredsize, around 100 μm could be produced. The viscosity of the chitosansolution depends on molecular weight and concentration. A 2.5% solutionof a low-molecular weight chitosan of 300 kDa produced good results. Itwas also discovered that these microbeads could be dried and reswelledwithout undergoing excessive deformation. The conditions used aredescribed in Example 4.

These microbeads also have a macroporous structure. This is indicatedfrom the bead density and also the partial transparency of themicrobead.

Examples of Microbead Production Processes:

Method 1: Emulsion/Solvent Evaporation

EXAMPLE 1 Low-Density Microbeads

Briefly, 200 mg of chitosan was completely dissolved in 11 ml of 0.1NHCl via manual mixing between 2 syringes connected with a luer-to-lueradapter, the solution was allowed to stand overnight to get rid of thebubbles. Meanwhile, 2% lecithin was dissolved in castor oil by heatingat 120° C. for 0.5 hour under magnetic stirring. Afterwards, the aqueousphase (8 g) was added into the oil phase (10 g) in a 50 ml beaker, theemulsification was performed at 400 rpm for 1.5 minutes with an overheadstirrer utilizing an anchor propeller.

Subsequently, the primary emulsion was quickly poured into a largeamount of an oil phase consisting of 20 g of light mineral oil and 20 gof corn oil, under constant magnetic stirring at 500 rpm. In order toallow the microbeads to solidify, the stirring was continued for 18hours at 28° C.

The oil phase containing microspheres was centrifuged at 1,000×g for 1min. The supernatant oil was decanted, and the micromicrobeads at thebottom were then washed 2 times with ethyl acetate, and 2 times withethanol by centrifugation to remove the oil and excess emulsifier. Theywere then neutralized by soaking in 5 M Na2CO3 solution for 10 minutes,washed with water to remove the salt on the surface, then they werewashed with ethanol, and air-dried.

The resultant chitosan microbeads can be stored under room condition inthe dry state, the content of chitosan in the wet microbeads is around10%.

EXAMPLE 2 High-Density Microbeads

Dissolve 300 mg of chitosan in 11 ml of 0.1N HCl via manual mixingbetween 2 syringes connected with a luer-to-luer adapter, the microbeadswere prepared using the same procedure in Example 1 except theemulsification speed was increased to 450 rpm. The obtainedmicromicrobeads have a higher solid content (˜15%).

EXAMPLE 3 BDDE Cross-Linked Medium-Density Microbeads

Preparation of ‘medium-density’ microbeads:

Dissolve 230 mg of chitosan in 11 ml of 0.1N HCl via overhead stirring,the microbeads were prepared using the same procedure in Example 1except the emulsification speed was decreased to 350 rpm for 2 minutes.The obtained microbeads have a medium solid content (˜14%) in theswelling state.

Cross-linking

First weigh around 280 mg of plain chitosan beads into 16 ml jacketedbeaker, then add 10 ml of 1% NaOH into this beaker, suspend the beadsunder mild magnetic stirring with a thin stirrer, keep continuous mixingfor 20 min. Second, 100 μl of BDDE was added into the beads suspension,the cross-linking reaction was allowed to proceed at 50° C. for 2 hours.Finally, collect the cross-linked beads, and then wash the beads with DIH2O to remove residual BDDE.

After drying, the resultant cross-linked chitosan microbeads can bestored under room conditions. The content of chitosan in the wetmicrobeads is around 14%.

Method 2: Emulsion/Neutralization Method

Example 4

While stirring 8.00 mL of castor oil with a small anchor paddle at 65rpm in a 20 mL beaker, 1.00 mL of 3% Chitopharm-S in 0.15 M HCl wereadded drop wise, avoiding hitting the paddle. The mixture was stirredfor 45 minutes and then the stir speed was increased to 100 rpm for 55minutes. The emulsion was neutralized by addition of concentrated NH₄OHat a rate of 50 microliters/hr over 30 minutes. The emulsion was thendiluted with ethanol and filtered several times to obtain the oil-freemicrobeads. Final washing with water and PBS, and resuspension in PBSresulted in the microbeads displayed in FIG. 4.

Cross-Linked HA Gel Phase Base for Chitosan Microbead Dermal Filler.

As noted, commercial HA-based dermal fillers such as Restylane®,Perlane®, Puragen®, the

Juvéderm® family, the Esthélis® family, and the Revanesse® family arebased on cross-linking of HA with ButaneDiolDiglycidylEther (BDDE). Thebasis of this technology was described in Laurent (1964) and in U.S.Pat. No. 4,716,154. Briefly, HA is dissolved in a strong base, such as1% NaOH solution, BDDE is added and the cross-linking takes place at anelevated temperature of approximately 50° C. An ether link is formed,primary at the C6 hydroxyl group. A schematic of the reaction is shownbelow.

After crosslinking, the gel that is formed is collected, milled to formgel particles, and purified before filling into syringes, and terminallysterilized by moist heat. A portion of unmodified HA can be included ornot, to alter somewhat the flow properties of the final product. Thoseskilled in the art can use this method to produce dermal fillers withexcellent characteristics of biocompatibility, softness, volumizingeffect, and durability.

The base continuous phase for the dermal filler system, includingmacroporous chitosan microbeads described herein, is the gel particlecomposition used in the product Revanesse® Ultra, manufactured byProllenium Medical Technologies, Inc. Milled, purified gel particles areavailable by the basic process described above. At that point, prior tosterilization, a microbead component can be added to the gel phase, andmixed thoroughly, for example in a double-planetary mixer, untildispersed uniformly in the gel. This composition can then be filled intosterile syringes, prior to loading in racks and terminally sterilized bymoist heat in an autoclave.

Given the size and softenss of the gel particles, and the size of thechitosan microbeads of approximately 100 μm, the overall composition canbe injected into the dermis with either a 27 G or 30 G needle or cannulaas demonstrated in our laboratory, and by technicians at the contractfacility carrying out the rat implant study.

Biological Response—Rat Implant Study

The biological response to our filler system, is of primary importancein terms of effectiveness. This can only be determined definitively inan animal implant study or in a human clinical trial. Prior to injectioninto a human subject, an animal study is usually performed. Inparticular, as we were interested in both safety from a toxicologicalviewpoint, and performance in terms of tissue response and durability.

An animal study provides strong evidence for both safety and performanceand to test the biological response of our chitosan bead/cross-linkedhyaluronic acid filler system, we conducted a study in Sprague Dawleyrats (Rattus norvegicus). This year-long study was conducted by ToxikonCorporation, a preclinical CRO in Bedford, Mass. from Dec. 31 2013 toDec. 30 2014. Chitosan beads from our solvent evaporation method, withtwo different mass densities of swelled chitosan microbeads were used,as well as a sample of medium-density beads cross-linked with BDDE.These bead samples were combined with BDDE-cross-linked gel particlesfrom a regular production lot of Revanesse Ultra and homogenized.Microbead concentrations were 25 mg/mL in all cases, HA concentrationwas also 25 mg/mL in all cases. The syringes were terminally-sterilizedin an autoclave.

Ten Sprague Dawley rats (rattus norvegicus) were selected for the study.Two animals were assigned to each of 5 time points for histopathologicalexamination: 1, 2, 8, 26, and 52 weeks. A volume of 0.2 mL of each ofthe three chitosan microbead/HA dermal filler test articles (high andlow density, and medium-density cross-linked microbeads), were implantedsubcutaneously into the back of each animal.

Two types of staining were used for the histopathology slides,hematoxylin and eosin (H&E), and Masson's trichrome staining, to bringout different features of the response. Generally, the trichromestaining was superior, for those characteristics in which we were mostinterested, in particular the evidence of stimulated collagendeposition. Collagen stains blue in the trichrome system and thedeposits were evident.

Results exceeded our expectations. A normal foreign-body response wasnoted. Even at 1 week infiltration of cells into the macroporous beadscould already be detected. In addition, there was no evidence of anexcessive inflammatory reaction at any of the time points, simply thenormal foreign-body reaction to the implant as a whole, clear depostionof collagen around the individual microbeads, as well as signficantcollagen deposition inside the macropores of the beads, due to ingressof fibroblasts into the microbead.

A large number of tissue samples were taken with photomicrographsprepared. Some of these are displayed in the Figures. Details arediscussed in the Description of the Figures:

DESCRIPTION OF THE FIGURES

FIG. 1 The ternary phase diagram for castor oil, corn oil, and lightmineral oil. Two coexistence curves are shown at temperatures of 20.7°C. and 26° C. Below these curves at the respective temperature, thesystem exists as two distinct phases, with different compositions. Abovethe curves a single phase exists, and all three components are misciblein this region. Experiments indicated that working close to thecoexistence curve was necessary for optimal conditions. Final samplesfor the animal implant study were prepared at an evaporation temperatureof 26° C., with the composition labeled as ‘1’ on the diagram (10/20/20for castor/corn/light mineral oils).

FIG. 2 Aqueous acidic chitosan droplets dispersed in castor oil in theprimary emulsion. Due to the high W/O ratio some castor oil is dispersedin the aqueous droplets forming an O/W/O emulsion. This is the source ofthe macroporous structure of the final chitosan microbeads.

FIG. 3 Displays a photomicrograph of three samples of chitosanmicrobeads, produced using Method 1, emulsion/solvent evaporation. Theseare the same samples used in the rat implant study. The size and shapeof the microbeads can be observed and in some of the beads the evidenceof the macroporous structure can be seen on the surface. The sizedistribution (volume-weighted), mean diameter, and standard deviationaround the mean are shown to the right of the photomicrograph. As can beseen the microbeads are nearly perfectly spherical. The mean diameter is˜95 μm±20 μm. As shown in FIG. 2, by adjusting the process conditions,beads of nearly identical size can be produced with signficantlydifferent chitosan mass densities, ranging from 9.9% to 16.2%.

FIG. 4 Displays a photomicrograph of a sample of chitosan microbeads,produced using Method 2, emulsion/neutralization. The size distributionis again shown to the right, with a mean diamter of 98 μm±??.

FIG. 5 Shows a result from experiments on in-vitro degradation of thedermal filler system, chitosan microbeads plus BDDE cross-linked HA gel.Bovine testicular hyaluronidase (BTH), and lysozyme were chosen asrepresentative of enzymes that will degrade HA and chitosan respectivelyin mammalian systems. The storage modulus of the dermal filler gel at afrequency of 1 Hz is measured to track the degradation. As shown,neither lysozyme nor BTH alone cause rapid degradation of the filler.However, lysozyme +BTH does cause a significant decrease in the storagemodulus. Finally, the base HA gel is shown to be degraded by BTH alone.This is an indication of an interaction between the HA gel and thechitosan beads. The interaction is the formation of a polyelectrolytecomplex at the surface of the beads.

FIG. 6 A more direct demonstration of the effect that the formation of apolyelectrolyte complex has on stabilizing the chitosan microbeadsagainst enzymatic degradation is shown here. The top twophotomicrographs show chitosan beads before and after exposure to aconcentrated lysozyme solution at 37° C., for 10 days in the presence ofHA gel. The bottom three photomicrographs show the effect ofconcentrated lysozyme on the same sample of chitosan microbeads, withoutthe presence of HA gel. After just 2 hours a bulk degradation of themicrobeads is evident, and after 3 days, and finally 10 days the beadshave lost most of their mass. It is seen that a rapid bulk degradationis occurring in the absence of HA.

FIG. 7 First histopathology slide showing overall reaction tosubcutaneous implant at 16×magnifications in the rat with low-densitybeads as an example. In all the histology slides shown trichromestaining was used. As noted, this brings out a feature of greatinterest, natural collagen depositon. On the left at 2 weeks, collagenlayer below muscle is visible (blue) but very little capsule formationon bottom of implant. On right at 52 weeks, a visible thin capsule canbe seen on the bottom of the implant, with no evidence of inflammation.

FIG. 8 At 63×magnification. These two photomicrographs demonstrate theresponse at 52 weeks, for cross-linked microbeads on the left andhigh-density microbeads on the right. In both cases there is clearevidence of collagen deposition around and on the surface of themicrobeads. The high-density microbeads on the right slso show clearevidence of collagen deposition within some of the beads. This is moreevident at higher magnification. Again there is no evidence of anyexcessive inflammatory reaction.

FIG. 9 In these photomicrographs at 400×magnification, evidence ofcollagen deposition within the microbeads is evident at 52 weeks. Thisis the case for low- and high-density microbeads as well as thecross-linked microbeads. Over additional time as the beads degrade thenatural collagen outside and within the bead will completely occupy thespace formerly taken up by the microbeads, leaving a natural collagenfilling effect where the original correction took place.

REFERENCES

Chitosan Reviews:

K. Kurita, ‘Chemistry and application of chitin and chitosan’, PolymerDegradation and Stability, 59 (1998) 117-120

D. K. Singh and A. R. Ray, Biomedical Applications of Chitin, Chitosan,and Their Derivatives, J.M.S.—Rev. Micromole. Chem. Phys., C 40(1),69-83 (2000)

E. Khor, ‘Review, Chitin: a biomaterial in waiting’, Current Opinion inSolid State and Materials Science 6 (2002) 313-317

M. N. V. Ravi Kumar, R. A. A. Muzzarelli, C. Muzzarelli, H. Sashiwa, andA. J. Domb, ‘Chitosan Chemistry and Pharmaceutical Perspectives’, Chem.Rev., 104, (2004) 6017-84

I. M. van der Lubben, J. C. Verhoef, G. Borchard, H. E. Junginger,‘Review, Chitosan and its derivatives in mucosal drug and vaccinedelivery’, European Journal of Pharmaceutical Sciences 14 (2001) 201-207

K. Kurita, ‘Chitin and Chitosan: Functional Biopolymers from MarineCrustaceans’, Marine Biotechnology, 8, 203-226 (2006)

R. C. F. Cheung, T. B. Ng, J. H. Wong and W. Y. Chan, ‘Review, Chitosan:An Update on Potential Biomedical and Pharmaceutical Applications’, Mar.Drugs 2015, 13, 5156-5186

I. Aranaz, M. Mengibar, R. Harris, I. Paños, B. Miralles, N. Acosta, G.Galed and A. Heras, ‘Functional Characterization of Chitin andChitosan’, Current Chemical Biology, (2009), 3, 203-230

Chitosan Microspheres

K.C. Gupta, F. H. Jabrail, ‘Effects of degree of deacetylation andcross-linking on physical characteristics, swelling and release behaviorof chitosan microspheres’, Carbohydrate Polymers 66 (2006) 43-54

A. J. Ribeiro, C. Silva, D. Ferreira, F. Veiga, ‘Chitosan-reinforcedalginate microspheres obtained through the emulsification/internalgelation technique’, European Journal of Pharmaceutical Sciences 25(2005) 31-40

V. R. Sinha, A. K. Singla, S. Wadhawan, R. Kaushik, R. Kumria, K.Bansal, S. Dhawan, ‘Review, □Chitosan microspheres as a potentialcarrier for drugs’, International Journal of Pharmaceutics 274 (2004)1-33

Y. Baimark and Y. Srisuwan, ‘Preparation of Polysaccharide-BasedMicrospheres □ by a Water-in-Oil Emulsion Solvent Diffusion Method forDrug Carriers’, International Journal of Polymer Science, Volume 2013,Article ID 761870,6 pages

Polyelectrolyte Complexes with HA

A. Denuziere, D. Ferrier, O. Damour, A. Domard, ‘Chitosan—chondroitinsulfate and chitosan—hyaluronate polyelectrolyte complexes: biologicalproperties’, Biomaterials, 19 (1998) 1275-1285

Y. Luo, Q. Wang, ‘Recent development of chitosan-based polyelectrolytecomplexes with natural polysaccharides for drug delivery’ InternationalJournal of Biological Macromolecules 64 (2014) 353-367

X. Deng, M. Cao, J. Zhang, K. Hu, Z. Yin, Z. Zhou, X. Xiao, Y. Yang, W.Sheng, Y. Wu, Y. Zeng, ‘Hyaluronic acid-chitosan nanoparticles forco-delivery of MiR-34a and doxorubicin in therapy against triplenegative breast cancer’, Biomaterials 35 (2014) 4333-4344

T. Funakoshi, ‘Novel chitosan-based hyaluronan hybrid polymer fibers asa scaffold in ligament tissue engineering’, Journal of BiomedicalMaterials Research Part A, Volume 74A, Issue 3, pages 338-346 (2005)

H. Lu and N. Hu, ‘Loading Behavior of {Chitosan/Hyaluronic Acid}nLayer-by-Layer Assembly Films toward Myoglobin: An ElectrochemicalStudy’, J. Phys. Chem. B 110, 23710-23718 (2006)

Wound Healing:

G. I. Howling , P. W. Dettmar, P. A. Goddard, F. C. Hampson, M. Dornish,E. J. Wood, ‘The effect of chitin and chitosan on the proliferation ofhuman skin fibroblasts and keratinocytes in vitro’, Biomaterials 22(2001) 2959-2966

K. Azuma, T. Osaki, S. Minami and Y. Okamoto, ‘Anticancer andAnti-Inflammatory Properties of Chitin and Chitosan Oligosaccharides’,J. Funct. Biomater, (2015), 6, 33-49

Biodegradability of Unmodified Chitosan

H. Kim, C. H. Tator, M. S. Shoichetl, ‘Chitosan implants in the ratspinal cord: Biocompatibility and biodegradation’, Journal of BiomedicalMaterials Research A, 97A(4), (2011) pp. 395-404.

J. Guzmán-Morales, C.-H. Lafantaisie-Favreau, G. Chen, C. D. Hoemann,‘Subchondral chitosan/blood implant-guided bone plate resorption andwoven bone repair is coupled to hyaline cartilage regeneration frommicrodrill holes in aged rabbit knees’, Osteoarthritis and Cartilage, 22(2014) pp. 323-333

Tissue Engineering:

C. D. Hoemann, J. Sun, A. Légaré, M. D. McKee and ⊐ M. D. Buschmann,‘Tissue engineering of cartilage using an injectable and adhesivechitosan-based cell-delivery vehicle’, OsteoArthritis and Cartilage(2005) 13, 318-329

In-Yong Kim, Seog-Jin Seo, Hyun-Seuk Moon, Mi-Kyong Yoo, In-Young Park,Bom-Chol Kim, Chong-Su Cho, ‘Research review paper ␣ Chitosan and itsderivatives for tissue engineering applications’, Biotechnology Advances26 (2008) 1-21

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A. Anithaa, S. Sowmya, P. T. S. Kumara, S. Deepthia, K. P. Chennazhia,H. Ehrlich, M. Tsurkanc, R. Jayakumara, ‘Chitin and chitosan in selectedbiomedical applications’, Progress in Polymer Science 39 (2014)1644-1667

L. Rami, S. Malaise, S. Delmond, J-C Fricain, R. Siadous, S. Schlaubitz,E. Laurichesse, J. Amédée, A. Montembault, L. David and L. Bordenave,‘Physicochemical modulation of chitosan-based hydrogels inducesdifferent biological responses: Interest for tissue engineering’,Journal of Biomedical Materials Research Part A, 102(10), (2014)3666-3676

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Cross-Linked HA Dermal Fillers:

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1-13. (canceled)
 14. A method for preparing unmodified chitosanmicrobeads by emulsification of an acidic chitosan solution into an oilphase in a primary emulsion containing an emulsifier, comprising thesteps of: forming a primary 0/W/O emulsion, diluting the primaryemulsion with two additional oils to form a secondary emulsion, with athree-component oil phase; and evaporating water from droplets of thechitosan solution to form solid microbeads with a macroporous structureafter oil removal.
 15. The method of claim 14, wherein the oil used inthe primary emulsion is castor oil.
 16. The method of claim 14, whereinthe emulsifier is chosen from one of: the hydrophobic Span® family, inparticular Sorbitan Monopalmitate and Sorbitan Monooleate; thehydrophilic Tween® family, particularly PEG-20 Sorbitan Isostearate;Castor oil derivatives such as PEG-40 Castor Oil, PEG-60 HydrogenatedCastor Oil, and Polyoxyl 35 Castor Oil; Glyceryl derivatives such asGlyceryl Palmitostearate, Glyceryl Oleate, Glyceryl Trioleate, andGlyceryl Laurate; the Poloxamer family of nonionic emulsifiers, inparticular Poloxamer 188; Hydrogenated Soybean Lecithin or Lecithin. 17.The method of claim 16, wherein the emulsifier is Lecithin at aconcentration of from 1% to 5%.
 18. The method of claim 14, wherein thetwo additional oils used to form the secondary emulsion are corn oil andlight mineral oil.
 19. The method of claim 14, wherein the secondaryemulsion contains castor, corn, and light mineral oil in a preferredratio of 10/20/20.
 20. The method of claim 14, wherein the chitosanmicrobeads are based on chitosan with a molecular weight of from 100 to2000 kDa.
 21. The method of claim 14, wherein the chitosan microbeadshave a degree of deacetylation from 65 to 95%.
 22. The method of claim14, wherein the acid is chosen from acetic acid, formic acid, adipicacid, ascorbic acid or lactic acid, or dilute inorganic acids such ashydrochloric acid or phosphoric acid.
 23. The method of claim 14,wherein hydrochloric acid concentrations are between 0.1N and 0.2N,Lactic acid concentrations are between 1 and 10, and Acetic acidconcentrations are between 2% and 10%.
 24. The method of claim 14,wherein the acidic chitosan solution consists of a chitosanconcentration between 1% and 5%.
 25. The method of claim 14, wherein theW/O ratio in the primary ratio is greater than 0.75.
 26. The method ofclaim 14, wherein the evaporation temperature is between 20° C. and 40°C.
 27. A cleaning, neutralization and drying process for preparingunmodified chitosin microbeads by emulsification of an acidic chitosansolution into an oil phase in a primary emulsion containing anemulsifier, said process comprising the steps of: forming a primary0/W/O emulsion, diluting the primary emulsion with two additional oilsto form a secondary emulsion, with a three-component oil phase;evaporating water from droplets of the chitosan solution to form solidmicrobeads with a macroporous structure after oil removal;centrifugation and isolation of the microbeads; washing with ethylacetate; repeating with n-hexane, and finally ethanol; neutralizing withsaturated Na₂C0₃; washing with DI water; equilibration withphosphate-buffered saline, until neutral pH achieved; and drying bydehydration with ethanol and air drying.
 28. A biocompatible, degradabledermal filler system, comprising unmodified macroporous chitosanmicrobeads dispersed uniformly in a continuous phase composed ofcross-linked hyaluronic acid gel particles and unmodified hylauronicacid, wherein the filler is loaded into pre-sterilized syringes, as partof the filler system.
 29. The system of claim 28, further comprising thepre-filled syringes being terminally sterilized by moist heat in anautoclave. 30-37. (canceled)